Plasmacytoid dendritic cells for treating cancer

Abstract

The present invention relates to the use of plasmacytoid dendritic cells for treating and / or preventing cancer.

Description

[Technical Field]

[0001] The present invention relates to the treatment and / or prevention of cancer. More specifically, the present invention relates to the use of plasmacytoid dendritic cells to treat and / or prevent cancer. [Background technology]

[0002] Cancer vaccine approaches that stimulate the immune system are one of the major strategies worldwide to overcome cancer. The primary cancer vaccine approach consists of priming and activating a patient's tumor-specific cytotoxic CD8+ T lymphocytes with dendritic cells (DCs), as these are professional antigen-presenting cells. However, cancer vaccines based on autologous DCs have proven difficult to produce and have shown little clinical benefit. Allogeneic DC-based vaccines have been proposed as an alternative to their autologous counterparts because they overcome several issues, including lack of reproducibility and the difficulty of production from patient blood. Furthermore, allogeneic responses resulting from the expression of mismatched HLA on allogeneic DCs are expected to generate activation stimuli that enhance the stimulation of antigen-specific CD8+ T cells (ASTCs).

[0003] In relation to melanoma, we previously demonstrated that plasmacytoid dendritic cells (PDCs) loaded with peptides derived from four melanoma-associated antigens activated rare tumor-specific antigen-specific antigens (ASTCs) present in the blood and, more importantly, in patients' tumors. These ASTCs proliferated, switched from a naive to a memory phenotype, exhibited cytotoxic potential, and killed autologous melanoma cells. Vaccination experiments in humanized mice also demonstrated the immunostimulatory and tumoricidal potential of PDC-based vaccines (Aspord C. et al., PLoS ONE, 2010). Furthermore, we reported a first-in-human Phase I / II trial in melanoma patients treated with PDC*-lineage cells loaded with peptides derived from four melanoma antigens (Clinical Trial Number NCT01863108) (Charles J. et al., OncoImmunology 2020). The product, named "GeniusVac-Mel4," was safe and well-tolerated with promising signs of clinical activity. Surprisingly, a significant increase in the frequency of circulating memory ASTC was observed, thereby demonstrating the priming and expansion of ASTC by the GeniusVac-Mel4 vaccine in humans.

[0004] However, this strategy appears to be suboptimal, and despite attempts by the scientific community to improve DC-based cancer vaccines, there is still a need to refine this strategy to enhance ASTC immune responses in patients in need. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Aspord C.et al, PLoS ONE, 2010 [Non-patent document 2] Charles J. et al, OncoImmunology 2020

[0006] Thus, there is an unmet medical need to improve these anti-cancer therapies. Summary of the Invention

[0007] The present invention relates to a method for treating and / or preventing cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a population of plasmacytoid dendritic cells (PDCs), wherein said therapeutically effective amount is at least 5,000,000 cells.

[0008] In some embodiments, the population of PDCs is contacted with at least one cancer antigen and / or fragment and / or variant thereof.

[0009] In some embodiments, the therapeutically effective amount of PDC is per cancer antigen and / or fragment and / or variant thereof.

[0010] In some embodiments, the at least one cancer antigen and / or fragment and / or variant thereof is selected from the group consisting of MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, CAMEL (CTAG2), GLULD1 (LGSN), HER2 (ERBB2), MUC1, survivin (BIRC5), NY-ESO-1, MULTI-MAGE, NY-BR-1, cyclin D1 (CCND1), PD -L1, CEA, EPCAM, IDO, LY-6K, MUC5AC and 5T4(TPBG), preferably the at least one cancer antigen and / or fragment and / or variant thereof is selected from the group comprising MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A9, MUC1, survivin (BIRC5), NY-ESO-1, CAMEL, MULTI-MAGE, 5T4(TPBG) and CCND1.

[0011] In some embodiments, the population of PDCs is administered at least 1 time, preferably at least 3 times, and more preferably at least 6 times, hi some embodiments, the population of PDCs is administered 1 to 10 times.

[0012] In some embodiments, the population of PDCs is administered intravenously, subcutaneously, intraarterially, or intramuscularly, preferably intravenously and / or subcutaneously.

[0013] In some embodiments, the cancer comprises or is selected from the group consisting of lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, squamous cell carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatic cancer, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, liver cancer, head and neck cancer, and the like.

[0014] In some embodiments, the cancer is lung cancer or melanoma. In some embodiments, the cancer is small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC). In some embodiments, the cancer is NSCLC.

[0015] In some embodiments, the method further comprises administering to the subject at least one additional anti-cancer agent.

[0016] In some embodiments, the methods further comprise administering at least one adjuvant to the subject.

[0017] In some embodiments, the method further comprises assessing the subject's immune background by blood sampling.

[0018] In some embodiments, the method further comprises assessing the immunogenicity of the subject by at least one blood sampling after vaccination.

[0019] The present invention also relates to a population of plasmacytoid dendritic cells (PDC) for use in the treatment and / or prevention of cancer, wherein the population of PDC is for administration or has been administered in a therapeutically effective amount of at least 5,000,000 cells.

[0020] The present invention further relates to a pharmaceutical composition comprising a population of plasmacytoid dendritic cells (PDCs) in a therapeutically effective amount of at least 5,000,000 cells for treating and / or preventing cancer in a subject in need thereof.

[0021] The present invention further relates to a kit comprising a therapeutically effective amount of a population of plasmacytoid dendritic cells (PDCs) of at least 5,000,000 cells for treating and / or preventing cancer in a subject in need thereof.

[0022] definition In the present invention, the following terms have the following meanings:

[0023] "About," when preceding a number, means plus or minus 10% of the value of said number.

[0024] "And / or" includes any and all possible combinations of one or more of the associated listed items, and when interpreted as an alternative ("or"), there are no combinations.

[0025] The terms "antibody" and "immunoglobulin" may be used interchangeably to refer to proteins having a combination of two heavy chains and two light chains, regardless of whether they have associated specific immunoreactivity. "Antibody" refers to such a collection of antibodies with significant known specific immunoreactivity to an antigen of interest (e.g., PD-1). The term "anti-PD-1 antibody" is used herein to refer to an antibody that exhibits immunological specificity for PD-1. Specificity for human PD-1 does not preclude cross-reactivity with other species. Antibodies and immunoglobulins comprise light and heavy chains, with or without interchain covalent bonds between them. Basic immunoglobulin structure in vertebrate systems is relatively well understood. The general term "immunoglobulin" encompasses five different classes of antibodies that can be biochemically distinguished. While the following discussion generally refers to the IgG class of immunoglobulin molecules, all five classes of antibodies are within the scope of the present invention. With regard to IgG, immunoglobulins comprise two identical light polypeptide chains with a molecular weight of approximately 23 kDa and two identical heavy chains with a molecular weight of approximately 53-70 kDa. The four chains are connected by disulfide bonds in a "Y" configuration, with the light chains supporting the heavy chains, which begin at the mouth of the "Y" and continue through the variable region. Antibody light chains are classified as either kappa (κ) or lambda (λ). Each heavy chain class may be associated with either a κ or λ light chain. Generally, when immunoglobulins are produced by either hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are covalently linked to each other, and the "tail" regions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chains, the amino acid sequences extend from the N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain. Those skilled in the art will understand that heavy chains are classified as gamma (γ), mu (μ), alpha (α), delta (Δ), or epsilon (ε), with several subclasses within each (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA IgD or IgE, respectively.Immunoglobulin subclasses or "isotypes" (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernible to those of skill in the art in light of the present disclosure and are therefore within the scope of the present invention. As noted above, the variable region of an antibody enables the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the light chain variable domain (VL domain) and heavy chain variable domain (VH domain) of an antibody combine to form a variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding sites present at the end of each arm of the "Y." The term "antibody" also extends to antibody mimetics.

[0026] As used herein, the term "antigen-binding fragment" refers to a portion or region of an antibody according to the present invention that contains fewer amino acid residues than the whole antibody. An "antigen-binding fragment" binds to an antigen and / or competes for antigen binding (e.g., specific binding to PD-1) with the whole antibody from which it is derived. Antibody antigen-binding fragments include, but are not limited to, single-chain antibodies, Fv, Fab, Fab', Fab'-SH, F(ab)'2, Fd, defucosylated antibodies, diabodies, triabodies, and tetrabodies.

[0027] "At least one" means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, 250, 500, 750, 10 3 ,10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 Including the above.

[0028] "Comprising," "comprises," and "comprised of" are used herein synonymously with "including," "includes," or "containing," and "contains," and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. These terms also encompass "consisting of."

[0029] "Epitope" refers to a specific arrangement of amino acids located on one or more proteins to which an antibody or antigen-binding fragment thereof or antibody mimetic binds. Epitopes often consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be linear (or sequence) or conformational, i.e., they include two or more sequences of amino acids from different regions of an antigen, which are not necessarily contiguous.

[0030] "Fc domain," "Fc portion," and "Fc region" refer to a C-terminal fragment of an antibody heavy chain, e.g., from about amino acid (aa) 230 to about aa 450 of a human gamma heavy chain, or corresponding sequences in other types of antibody heavy chains (e.g., α, δ, ε, and μ of human antibodies), or naturally occurring allotypes thereof.

[0031] "Individual" or "subject" refers to an animal, preferably a mammal, more preferably a human. In one embodiment, the individual is male. In another embodiment, the individual is female. In one embodiment, the individual may be a "patient," i.e., a warm-blooded animal, more preferably a human, awaiting or receiving medical care, or having been / is / will be the subject of medical treatment, and being monitored for the development of cancer. In one embodiment, the individual is an adult (e.g., a subject over the age of 18). In another embodiment, the individual is a child (e.g., a subject under the age of 18).

[0032] "Polypeptide" refers to any peptide or protein containing two or more amino acids linked together by peptide bonds or modified peptide bonds, i.e., peptide isosteres, thereby forming a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, pegylation, or any other manipulation, such as conjugation with a labeling component. As used herein, the term "amino acid" includes natural and / or unnatural or synthetic amino acids, including glycine and both D- and L-enantiomers, as well as amino acid analogs and peptidomimetics. "Polypeptide" refers to both short chains, oligopeptides, or oligomers, commonly referred to as "peptides," and longer chains, commonly referred to as "proteins." Therefore, within the scope of the present invention, the terms "peptide," "polypeptide," and "protein" are used interchangeably. Polypeptides may contain amino acids other than the 20 genetically encoded amino acids.

[0033] "Preventing cancer" is intended to mean avoiding the occurrence of at least one adverse effect or symptom of cancer.

[0034] "Treating cancer" or "treatment" or "palliative" refers to both therapeutic treatment and prophylactic or preventative measures, the goal being to prevent or slow (alleviate) cancer. Those in need of treatment include those already with cancer, as well as those prone to have cancer or those in whom cancer must be prevented. After an individual or mammal receives a therapeutic amount of a polypeptide according to the present invention, the "treatment" for cancer is successful if the individual exhibits an observable and / or measurable reduction in or absence of one or more of the following: a reduction in the number of cancer cells; a reduction in the percentage of total cells that are cancerous; and / or some alleviation of one or more symptoms associated with cancer; a reduction in morbidity and mortality, and an improvement in quality of life issues. The above parameters for assessing successful treatment and improvement of cancer can be readily measured by routine procedures familiar to physicians.

[0035] A "therapeutically effective amount" is intended to refer to a level or amount of an agent intended to (1) delay or prevent the onset of cancer; (2) slow or stop the progression, progression, or worsening of one or more symptoms of cancer; (3) bring about an improvement in the symptoms of cancer; (4) reduce the severity or incidence of cancer; or (5) prevent cancer formation, without causing significant negative or harmful side effects to the target. In one embodiment, a therapeutically effective amount is administered prior to the onset of cancer formation in the case of prophylactic or preventative treatment. In a particular embodiment, the cancer is lung cancer.

[0036] "Vaccine" refers to any preparation containing a substance or group of substances intended to make the subject's immune system respond to a given antigen, especially tumors.Prophylactic vaccines are used to prevent subjects from having a specific disease, especially cancer, or simply to alleviate the symptoms of disease, especially cancer.Therapeutic vaccines are intended to treat a specific disease, especially cancer, in subjects.Anti-cancer vaccines contain one or more tumor antigens that induce an immune response against tumor cells.

[0037] A "variant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another reference polynucleotide. Changes in the nucleotide sequence of a variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes can result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence, as described below. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions (preferably conservative), additions, or deletions, in any combination. Substituted or inserted amino acid residues may or may not be those encoded by the genetic code. A variant of a polynucleotide or polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis. Variants should retain one or more of the biological activities of the reference polypeptide. DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention relates to a method for treating and / or preventing cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a population of plasmacytoid dendritic cells (PDCs), wherein said therapeutically effective amount is at least 5,000,000 cells.

[0039] In some embodiments, the methods according to the present invention are for vaccination purposes.

[0040] Plasmacytoid dendritic cells (PDCs) are a subset of dendritic cells that are, among other things, producers of type I interferon and initiators of immune responses, particularly anti-tumor immune responses.

[0041] Methods for isolating, culturing, and using PDCs are published in Chaperot et al., Journal of Immunology vol. 176, 1 (2006): 248-55; Lui et al., PloS one vol. 4, 9 e7111.22 Sep. 2009; Aspord et al., The Journal of Investigative Dermatology vol. 132, 10 (2012): 2395-2406; and Hannani et al., International Journal of Molecular Sciences, 2023, 24 (3), 1897, which are incorporated herein by reference.

[0042] In some embodiments, the PDCs are cultured under suitable conditions. Methods for culturing cells in vitro are well known in the art and are routinely practiced.

[0043] In some embodiments, the PDC is maintained in a controlled atmosphere, ie, controlled temperature, pressure, and composition, typically 37° C., 1 atm, 5% CO 2 .

[0044] In some embodiments, PDCs are cultured in a substrate or vessel. In some embodiments, the substrate is plastic. In some embodiments, the plastic substrate is a vessel or container suitable for cell culture, typically a plastic flask or plastic dish.

[0045] In one embodiment, PDCs are plated onto a substrate that allows cell adhesion. In certain embodiments, the plastic substrate is treated with at least one agent that promotes cell adhesion, survival, and / or proliferation. Illustratively, in some embodiments, the plastic substrate is treated with poly-D-lysine, gelatin, and / or collagen. In some embodiments, the agent forms a layer or matrix on the surface of the plastic substrate.

[0046] In another embodiment, the PDCs are maintained in a substrate that prevents cell adhesion. In some embodiments, the PDCs are cultured in suspension.

[0047] In some embodiments, PDCs are cultured in a culture medium that sustains their further proliferation, generally a liquid culture medium that may contain serum or may be serum-free.

[0048] In some embodiments, PDCs are grown in suspension in a defined medium in a bioreactor.

[0049] Culture media are known in the art. The term "culture medium" or "cell culture medium" or "medium" refers to an aqueous liquid or gelatinous substance containing nutrients that can be used to maintain or grow cells. Cell culture media may contain serum or may be serum-free. These media, which may be further supplemented with an appropriate mixture of organic or inorganic compounds, can promote the growth / adhesion or elimination / detachment of specific cell types in addition to providing nutrients and / or growth promoters.

[0050] In some embodiments, the culture medium comprises basal medium formulations known in the art, including, but not limited to, Eagle's Minimum Essential Medium (MEM), OPTI-MEM, Dulbecco's Modified Eagle's Medium (DMEM), alpha modified minimal essential medium (alpha-MEM), Basal Medium Essential (BME), Iscove's Modified Dulbecco's Medium (IMDM), BGJb Medium, F-12 Nutrient Mixture (Ham), Liebovitz L-15, DMEM / F-12, Essential Modified Eagle's Medium (EMEM), RPMI-1640, 199 Medium, Waymouth's MB 752 / 1 X-VIVO-15, or Williams Medium E, as well as modifications and / or combinations thereof. The compositions of the above basal media are generally known in the art, and it is within the skill of one of ordinary skill in the art to modify or adjust the concentrations of the media and / or media supplements as needed to culture PDCs.

[0051] In a preferred embodiment, the culture medium used to culture the population of PDCs is X-VIVO-15 or a variant thereof.

[0052] By way of example and not limitation, these components may include inorganic salts (particularly salts containing Na, K, Mg, Ca, Cl, P, and optionally Cu, Fe, Se, and Zn), physiological buffers (e.g., HEPES, bicarbonate), nucleotides, nucleosides and / or nucleic acid bases, ribose, deoxyribose, amino acids, vitamins, antioxidants (e.g., glutathione), and carbon sources (e.g., glucose, pyruvates, e.g., sodium pyruvate, acetates, e.g., sodium acetate), etc. It will also be apparent that many media are available as low-glucose formulations, with or without sodium pyruvate.

[0053] In some embodiments, the basal medium is supplemented with one or more additional components selected from the group consisting of transferrin, selenium salts, amino acids, sugars, and combinations thereof. These components can be included in salt solutions, such as, but not limited to, Hank's Balanced Salt Solution (HBSS) and Earle's Salt Solution. Additional antioxidant supplements, such as β-mercaptoethanol, may also be added. Many basal media already contain amino acids, but some amino acids, such as L-glutamine, which are known to be less stable in solution, can be supplemented later.

[0054] In some embodiments, the culture medium is further supplemented with antibiotic and / or antifungal compounds, such as typically a mixture of penicillin and streptomycin, and / or other compounds such as, but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, zeocin, and combinations thereof.

[0055] In some embodiments, the culture medium is further supplemented with a fungicide compound.

[0056] In some embodiments, the culture medium is further supplemented with a hormone selected from the group consisting of D-aldosterone, diethylstilbestrol (DES), dexamethasone, estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin / human growth hormone (HGH), thyrotropin, thyroxine, L-thyronine, epidermal growth factor (EGF), human recombinant epidermal growth factor, and combinations thereof. The final concentration of the hormone can range from 0.01 to 1000 ng / mL, preferably 0.1 to 100 ng / mL, and more preferably 1 to 10 ng / mL.

[0057] In some embodiments, the culture medium is further supplemented with lipids and lipid carriers selected from the group including or consisting of cyclodextrin, cholesterol, linoleic acid conjugated to albumin, linoleic acid and oleic acid conjugated to albumin, unconjugated linoleic acid, linoleic-oleic-arachidonic acid conjugated to albumin, unconjugated oleic acid and oleic acid conjugated to albumin, etc. Albumin can be used in fatty acid-free formulations as well.

[0058] In some embodiments, the culture medium is further supplemented with mammalian plasma or serum. In some embodiments, the concentration of plasma or serum in the culture medium is 0.01% to 20%, preferably 0.1% to 10%, and more preferably 1% to 5%. In some embodiments, the concentration of plasma or serum in the culture medium is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.

[0059] The term "serum" is conventionally defined as being obtained from a sample of whole blood by first inducing coagulation in the sample, followed by separating the clot and cellular components of the blood sample thus formed from the liquid component (serum) by an appropriate technique, typically by centrifugation. An inert catalyst, such as glass beads or powder, can promote coagulation. Advantageously, serum can be prepared using a serum separator (SST) containing a catalyst inert to mammals. Plasma or serum often contains cellular elements and components necessary for survival and growth.

[0060] In some embodiments, plasma or serum for use in the media described herein may comprise human plasma or serum, or plasma or serum derived from a non-human animal, preferably a non-human mammal, such as non-human primate (e.g., lemur, monkey, ape), fetal or adult cow, horse, pig, lamb, goat, dog, rabbit, mouse, or rat serum or plasma, etc. In some embodiments, the culture medium is further supplemented with a serum replacement or analog.

[0061] As will be appreciated by those skilled in the art, cultured PDCs can be counted to facilitate replating of PDCs at a desired density.

[0062] In some embodiments, the PDC is 10 0 ~10 5 cells / cm 2 PDCs are cultured by replanking them at a density of 1 / 100 to 1 / 2 at a passage ratio of approximately 1 / 100 to 1 / 2. As used herein, passage ratio refers to the fraction of passaged PDCs seeded into an empty (typically new) culture vessel of the same surface area and / or volume as the vessel from which the PDCs were obtained.

[0063] The type of culture vessel and the type of surface and / or volume that allows for PDC growth into the culture vessel and cell culture medium may be the same as that originally used and described above herein, or may be different.

[0064] In some embodiments, the population of PDCs is free of contamination by bacteria, fungi, protozoa, archaea, or other microbial species.

[0065] In some embodiments, the PDC is stored for future use.

[0066] In some embodiments, PDCs are cryopreserved for storage. In some embodiments, PDCs are stored at temperatures between -20°C and -200°C, preferably between -80°C and -200°C. In some embodiments, PDCs are stored at approximately -196°C, typically with the cells stored in liquid nitrogen. In some embodiments, PDCs are "flash frozen" in liquid nitrogen prior to storage.

[0067] In some embodiments, the PDCs are contacted with at least one cryoprotectant prior to cryopreservation. Cryoprotectants are known in the art and include, for example, glycerol, sucrose, serum, etc.

[0068] In some embodiments, the population of PDCs is characterized in that it is substantially pure.

[0069] In some embodiments, a substantially pure population of PDCs contains 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001% or less of other contaminating cell types.

[0070] In some embodiments, a substantially pure population of PDCs contains 0% of other contaminating cell types. In some embodiments, a substantially pure cell population contains no more than one cell type.

[0071] In a preferred embodiment, the population of PDCs is clinical grade.

[0072] In some embodiments, the population of PDCs is irradiated prior to administration to a human. In some embodiments, the irradiation dose is 10-100 Gy. In preferred embodiments, the irradiation dose is 30-60 Gy.

[0073] In some embodiments, the PDCs express human-specific major histocompatibility complex (MHC) antigens.

[0074] In some embodiments, the PDC expresses class I MHC, e.g., human leukocyte antigen (HLA). In some embodiments, the HLA comprises or consists of HLA-A, HLA-B, and / or HLA-C subtypes.

[0075] In some embodiments, HLA is a heterodimer, composed of a heavy alpha chain and a smaller beta chain. In some embodiments, the alpha chain is encoded by a variant HLA-A gene, generating the HLA-A serotype group. In some embodiments, the HLA serotype is selected from the list including or consisting of HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:05, HLA-A*02:06, HLA-A*02:11.

[0076] In a specific embodiment, the PDCs express HLA-A. In a preferred embodiment, the PDCs express the HLA-A2 subtype. In a more preferred embodiment, the PDCs express the HLA-A*02:01 serotype.

[0077] In some embodiments, the PDCs are genetically modified.

[0078] As used herein, "genetically modified" means that the genome of the PDC contains at least one modification consisting of a mutation and / or the insertion of one or more nucleic acid sequences. Methods for genetically modifying cells are known in the art and include non-mimetic techniques such as CRIPR-Cas-based techniques, zinc finger nuclease-based techniques, TALENs, and the like. In a preferred embodiment, the PDC is constitutively modified, i.e., the genomic modification is transmitted to daughter cells; typically, the PDC is transduced by methods known in the art, such as lentiviral vectors. In another, less preferred embodiment, the PDC is transiently modified; typically, the PDC is transfected.

[0079] In some embodiments, the PDCs are genetically modified to express at least one protein of interest.

[0080] As used herein, "at least one" means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19 or 20.

[0081] As used herein, "genetically modified to express at least one protein of interest" means that the PDC expresses at least one protein of interest or its encoding transcript at 1.2-fold, 1.5-fold, 2-fold, 3-fold, 5-fold, 10-fold or more compared to unmodified PDC. In some embodiments, the PDC overexpresses at least one protein of interest.

[0082] In some embodiments, at least one protein of interest is a secreted protein or a surface protein.

[0083] In some embodiments, at least one protein of interest is a secreted protein. In some embodiments, the secreted protein is a cytokine. In some embodiments, the cytokine is an interleukin. In some embodiments, the interleukin is selected from the group including or consisting of IL-2, IL-7, IL-12, and IL-15. In some embodiments, the interleukin is IL-12 or IL-15.

[0084] In some embodiments, the at least one protein of interest is IL-12.

[0085] In humans, IL-12 typically comprises two subunits, IL12A (or p35) and IL12B (or p40), which typically have the sequences of Uniprot Ref. P29459 and Uniprot Ref. P29460, respectively, which are incorporated herein by reference. IL-12 is referred to interchangeably as IL-12, IL12, interleukin-12, and p70.

[0086] In some embodiments, the at least one protein of interest is IL-15.

[0087] In humans, IL-15 typically has the sequence as disclosed in Uniprot Ref. P40933, which is incorporated herein by reference. IL-15 is referred to interchangeably as IL-15, IL15, and interleukin 15.

[0088] In some embodiments, the at least one protein of interest is IL-7.

[0089] In humans, IL-7 typically has the sequence as disclosed in Uniprot Ref. P13232, which is incorporated herein by reference. IL-7 is referred to interchangeably as IL-7, IL7, and interleukin 7.

[0090] In some embodiments, at least one protein of interest is a surface protein.

[0091] In some embodiments, the surface protein is from the B7 family of proteins. In some embodiments, the surface protein from the B7 family of proteins is CD80 or CD86.

[0092] In some embodiments, the at least one protein of interest is CD80.

[0093] In humans, CD80 typically has the sequence as disclosed in Uniprot Ref. P33681, which is incorporated herein by reference. CD80 is referred to interchangeably as CD80, B7, B7-1, B7.1, B1, CD28LG, CD28LG1, and LAB7.

[0094] In some embodiments, the at least one protein of interest is CD86.

[0095] In humans, CD86 typically has the sequence as disclosed in Uniprot Ref. P42081, which is incorporated herein by reference. CD86 is also referred to interchangeably as CD86, B7-2, B7.2, B70, CD28LG2, and LAB72.

[0096] In some embodiments, at least one protein of interest is mutated, hi some embodiments, at least one protein of interest comprises at least one amino acid mutation compared to a wild-type protein of interest.

[0097] As used herein, "at least one" means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20. In some embodiments, "amino acid mutations" include substitutions, deletions, insertions, inversions, and combinations thereof.

[0098] In some embodiments, the mutation in the at least one protein of interest does not have a deleterious effect on PDCs, i.e., the mutation in the at least one protein of interest does not alter the viability and / or proliferation ability of PDCs. In some embodiments, the mutation in the at least one protein of interest is not oncogenic.

[0099] In some embodiments, mutation of at least one protein of interest increases the efficiency of PDCs as antigen-presenting cells by at least 1.5-fold, 2-fold, 3-fold, or more, hi some embodiments, mutation of at least one protein of interest increases the survival and / or circulation time of PDCs by at least 1.5-fold, 2-fold, 3-fold, or more.

[0100] In some embodiments, the PDCs are genetically modified to reduce (knock down) or eliminate (knock out) expression of at least one protein or the gene encoding it.

[0101] As used herein, "reduce" refers to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, and up to 99% reduction in expression from basal expression of at least one protein or gene encoding it. As used herein, "reduce" refers to a 100% reduction in expression from basal expression of at least one protein or gene encoding it.

[0102] As antigen-presenting cells, PDCs can activate naive and memory T cells into activated antigen-specific T cells either by direct priming or by cross-priming (i.e., presentation of exogenously derived antigens). This second mechanism allows PDCs to be contacted, loaded, pulsed, primed, etc. with any given antigen or combination of antigens of interest. The loaded PDCs then activate T cells specific for the antigen or combination of antigens of interest.

[0103] In some embodiments, the population of PDCs is contacted with at least one cancer antigen and / or fragment and / or variant thereof, hi some embodiments, the population of PDCs is contacted with at least two, three, or four cancer antigens and / or fragments and / or variants thereof.

[0104] As used herein, the term "contacting" is intended to mean that a population of PDCs is incubated with one or several cancer antigens and / or fragments and / or variants thereof, resulting in "loading" of the PDC population with one or several cancer antigens and / or fragments and / or variants thereof. Within the scope of the present invention, the terms "contacting," "loading," or "incubating" can be used interchangeably. This incubation itself is referred to herein interchangeably as the "loading step."

[0105] In some embodiments, incubation is carried out in a culture medium described herein or in a biological buffer, preferably in a culture medium, more preferably in X-VIVO-15 culture medium.

[0106] In some embodiments, the incubation is carried out at a temperature between 32°C and 42°C, preferably 37°C.

[0107] In some embodiments, the incubation is for a period of 30 minutes to 24 hours.

[0108] In some embodiments, the incubation is for a period of 1 hour to 24 hours, 2 hours to 24 hours, 4 hours to 24 hours, 8 hours to 24 hours, or 16 hours to 24 hours.

[0109] In some embodiments, the incubation is for a period of 30 minutes to 16 hours, 30 minutes to 8 hours, 30 minutes to 4 hours, or 30 minutes to 2 hours.

[0110] In some embodiments, the incubation is for a period of 1 hour to 16 hours, 2 hours to 8 hours, or 2 hours to 4 hours.

[0111] In a preferred embodiment, the incubation is for about 3 hours.

[0112] In some embodiments, the cell concentration of the population of PDCs used in the loading step is 1 x 10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , or 1 × 10 7 In some embodiments, the PDC cell concentration is 1 x 10 4 ~1×10 8 In a preferred embodiment, the PDC cell concentration is about 1 x 10 cells / mL. 6 cells / mL.

[0113] In some embodiments, the concentration of the cancer antigen and / or fragment and / or variant thereof used in the loading step is comprised between 1 μM and 10 μM final concentration, in some embodiments, the concentration of the cancer antigen and / or fragment and / or variant thereof used in the loading step is comprised between 2 μM and 5 μM final concentration.

[0114] In some embodiments, β2-microglobulin is also added to the loading step.

[0115] In some embodiments, the concentration of β2-microglobulin used in the loading step is in the range of 0.1 to 1 μg / mL. In a preferred embodiment, the concentration of β2-microglobulin is about 0.1 μg / mL.

[0116] In some embodiments, the population of PDCs is irradiated after the loading step. In some embodiments, the irradiation dose is 10-100 Gy. In preferred embodiments, the irradiation dose is 30-60 Gy.

[0117] In some embodiments, the population of PDCs is substantially free of free cancer antigens and / or fragments and / or variants thereof, i.e., about 100% of the cancer antigens and / or fragments and / or variants thereof is loaded onto the population of PDCs.

[0118] In some embodiments, the at least one cancer antigen and / or fragment and / or variant thereof is not expressed in healthy lung tissue. In some embodiments, the at least one cancer antigen and / or fragment and / or variant thereof has a low expression level in healthy tissue, preferably healthy lung tissue.

[0119] In some embodiments, the at least one cancer antigen and / or fragment and / or variant thereof is a molecule selected from the group comprising or consisting of a peptide, a polypeptide, a protein, a polysaccharide, a lipid, a nucleic acid, and combinations thereof.

[0120] In some embodiments, the at least one cancer antigen and / or fragment and / or variant thereof is a molecule selected from the group comprising or consisting of a peptide, a polypeptide, and a protein.

[0121] In preferred embodiments, the at least one cancer antigen and / or fragment and / or variant thereof is a peptide or polypeptide comprising at least 3, at least 5, at least 10, at least 15, at least 20, or more amino acid residues.

[0122] In some embodiments, the peptide comprises natural and unnatural amino acids. In preferred embodiments, the peptide comprises or consists of natural amino acids. In other embodiments, the peptide comprises or consists of unnatural amino acids.

[0123] In some embodiments, the peptide comprises at least one post-translational modification on at least one amino acid residue, hi some embodiments, the peptide is glycosylated, glycated, phosphorylated, biotinylated, oxidized, nitrated, nitrosylated, acylated, alkylated, acetylated, methylated, lipidated, ubiquitinated, carboxylated, pegylated, or a combination thereof.

[0124] In another embodiment, the at least one cancer antigen and / or fragment and / or variant thereof is a polysaccharide. In another embodiment, the at least one cancer antigen and / or fragment and / or variant thereof is a lipid. In another embodiment, the at least one cancer antigen and / or fragment and / or variant thereof is a nucleic acid molecule.

[0125] In some embodiments, at least one cancer antigen and / or its fragment and / or variant induces an immune response, i.e., at least one cancer antigen and / or its fragment and / or variant is immunogenic. In preferred embodiments, at least one cancer antigen and / or its fragment and / or variant induces an adaptive immune response. In further preferred embodiments, at least one cancer antigen and / or its fragment and / or variant induces a T-cell immune response.

[0126] In some embodiments, the at least one cancer antigen and / or fragment and / or variant thereof comprises or is selected from the group consisting of MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, CAMEL (CTAG2), GLULD1 (LGSN), HER2 (ERBB2), MUC1, survivin (BIRC5), NY-ESO-1, MULTI-MAGE, NY-BR-1, cyclin D1 (CCND1), PD-L1, CEA, EPCAM, IDO, LY-6K, MUC5AC, and 5T4 (TPBG).

[0127] In a preferred embodiment, the at least one cancer antigen and / or fragment and / or variant thereof comprises or is selected from the group consisting of MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A9, MUC1, survivin (BIRC5), NY-ESO-1, CAMEL, MULTI-MAGE, 5T4 (TPBG) and CCND1. In a preferred embodiment, the at least one cancer antigen and / or fragment and / or variant thereof comprises or is selected from the group consisting of MAGE-A1, MAGE-A3, MAGE-A4, MUC1, survivin (BIRC5), NY-ESO-1 and MULTI-MAGE. In a preferred embodiment, the at least one cancer antigen and / or fragment and / or variant thereof comprises or is selected from the group consisting of MAGE-A3, MUC1, survivin (BIRC5) and NY-ESO-1.

[0128] In another embodiment, the at least one cancer antigen and / or fragment and / or variant thereof comprises or is selected from the group consisting of NY-ESO-1, CAMEL, MAGE-A2, MAGE-A3, and MAGE-A9.

[0129] In some embodiments, the at least one cancer antigen and / or fragment and / or variant thereof is MAGE-A1. In some embodiments, the at least one cancer antigen and / or fragment and / or variant thereof is MAGE-A3. In some embodiments, the at least one cancer antigen and / or fragment and / or variant thereof is MAGE-A4. In some embodiments, the at least one cancer antigen and / or fragment and / or variant thereof is MAGE-A9. In some embodiments, the at least one cancer antigen and / or fragment and / or variant thereof is MUC1. In some embodiments, the at least one cancer antigen and / or fragment and / or variant thereof is survivin (BIRC5). In some embodiments, the at least one cancer antigen and / or fragment and / or variant thereof is NY-ESO-1. In some embodiments, the at least one cancer antigen and / or fragment and / or variant thereof is CAMEL (CTAG2). In some embodiments, the at least one cancer antigen and / or fragment and / or variant thereof is MULTI-MAGE. In some embodiments, the at least one cancer antigen and / or fragment and / or variant thereof is 5T4 (TPBG). In some embodiments, the at least one cancer antigen and / or fragment and / or variant thereof is CCND1.

[0130] Methods according to the invention comprise administering a therapeutically effective amount of a population of PDCs according to the invention. The meaning of the expression "therapeutically effective dose" within the scope of the invention is explained in more detail below.

[0131] In one embodiment, the population of PDCs is administered at a dose of at least 5,000,000, at least 6,000,000, at least 7,000,000, at least 8,000,000, at least 9,000,000, or at least 10,000,000 cells. In some embodiments, the population of PDCs is administered at a dose of at least 15,000,000, at least 20,000,000, at least 30,000,000, at least 40,000,000, at least 50,000,000, at least 60,000,000, at least 70,000,000, at least 80,000,000, at least 90,000,000, or at least 100,000,000 cells. In some embodiments, the population of PDCs is administered at a dose of at least 120,000,000, at least 130,000,000, at least 140,000,000, or at least 150,000,000 cells.

[0132] In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 150,000,000 cells. In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 140,000,000 cells. In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 120,000,000 cells. In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 100,000,000 cells. In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 80,000,000 cells. In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 70,000,000 cells. In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 60,000,000 cells. In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 50,000,000 cells. In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 30,000,000 cells. In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 20,000,000 cells. In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 25,000,000 cells. In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 20,000,000 cells.

[0133] In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 150 million cells. In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 140 million cells. In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 120 million cells. In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 100 million cells. In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 80 million cells. In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 70 million cells. In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 60 million cells. In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 50 million cells. In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 30 million cells. In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 25 million cells. In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 20 million cells. In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 15 million cells.

[0134] In one embodiment, the population of PDCs is administered at a dose of about 20 million to about 150 million cells. In one embodiment, the population of PDCs is administered at a dose of about 20 million to about 140 million cells. In one embodiment, the population of PDCs is administered at a dose of about 20 million to about 120 million cells. In one embodiment, the population of PDCs is administered at a dose of about 20 million to about 100 million cells. In one embodiment, the population of PDCs is administered at a dose of about 20 million to about 80 million cells. In one embodiment, the population of PDCs is administered at a dose of about 20 million to about 70 million cells. In one embodiment, the population of PDCs is administered at a dose of about 20 million to about 60 million cells. In one embodiment, the population of PDCs is administered at a dose of about 20 million to about 50 million cells. In one embodiment, the population of PDCs is administered at a dose of about 20 million to about 30 million cells.

[0135] In one embodiment, the dose as described herein above refers to the dose of cells contacted with at least one cancer antigen. In some embodiments, the population of PDCs is contacted with at least two or more cancer antigens and / or fragments and / or variants thereof. Thus, in some embodiments, the dose of cells administered is the dose per cancer antigen loaded onto the cells.

[0136] In one embodiment, the population of PDCs is administered at a dose of at least 5,000,000, at least 6,000,000, at least 7,000,000, at least 8,000,000, at least 9,000,000, or at least 10,000,000 cells per cancer antigen and / or fragment and / or variant thereof. In some embodiments, the population of PDCs is administered at a dose of at least 15,000,000, at least 20,000,000, at least 30,000,000, at least 40,000,000, at least 50,000,000, at least 60,000,000, at least 70,000,000, at least 80,000,000, at least 90,000,000, or at least 100,000,000 cells per cancer antigen and / or fragment and / or variant thereof. In some embodiments, the population of PDCs is administered at a dose of at least 120,000,000, at least 130,000,000, at least 140,000,000, or at least 150,000,000 cells per cancer antigen and / or fragment and / or variant thereof.

[0137] In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 150,000,000 cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 140,000,000 cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 120,000,000 cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 100,000,000 cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 80,000,000 cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 70,000,000 cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 60,000,000 cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 50,000,000 cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 30,000,000 cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 20,000,000 cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 25,000,000 cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 5,000,000 to about 20,000,000 cells per cancer antigen and / or fragment and / or variant thereof.

[0138] In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 150 million cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 140 million cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 120 million cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 100 million cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 80 million cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 70 million cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 60 million cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 50 million cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 30 million cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 25 million cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 20 million cells per cancer antigen and / or fragment and / or variant thereof.In one embodiment, the population of PDCs is administered at a dose of about 10 million to about 15 million cells per cancer antigen and / or fragment and / or variant thereof.

[0139] In one embodiment, the population of PDCs is administered at a dose of about 20 million to about 150 million cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 20 million to about 140 million cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 20 million to about 120 million cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 20 million to about 100 million cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 20 million to about 80 million cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 20 million to about 70 million cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 20 million to about 60 million cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 20 million to about 50 million cells per cancer antigen and / or fragment and / or variant thereof. In one embodiment, the population of PDCs is administered at a dose of about 20 million to about 30 million cells per cancer antigen and / or fragment and / or variant thereof.

[0140] In another embodiment, the population of PDCs is administered at a dose of 1,000 to 1,000,000 cells per kg of body weight.

[0141] In some embodiments, the population of PDCs is administered at a dose of 5,000 to 1,000,000 cells per kg of body weight, 10,000 to 1,000,000 cells per kg of body weight, or 50,000 to 1,000,000 cells per kg of body weight.

[0142] In some embodiments, the population of PDCs is administered at a dose of 10,000-500,000 cells per kg of body weight, 10,000-400,000 cells per kg of body weight, or 10,000-300,000 cells per kg of body weight.

[0143] In some embodiments, the population of PDCs is administered at a dose of 20,000-500,000 cells / kg body weight, 30,000-400,000 cells / kg body weight, 50,000-300,000 cells / kg body weight, or 200,000-300,000 cells / kg body weight. In some embodiments, the population of PDCs is administered at a dose of about 280,000 cells / kg body weight.

[0144] Methods for cell counting are known in the art and include, but are not limited to, hemocytometers, automated cell counters, flow cytometers, spectrophotometers, and the like.

[0145] In one embodiment, the population of PDCs is administered at least once. In a preferred embodiment, the population of PDCs is administered at least three times. In a more preferred embodiment, the population of PDCs is administered at least six times.

[0146] Within the scope of the present invention, the expression "at least once" is intended to mean at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, or more. Within the scope of the present invention, the expression "at least 3 times" is intended to mean at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, or more. Within the scope of the present invention, the expression "at least 6 times" is intended to mean at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, or more.

[0147] In some embodiments, the population of PDCs is administered 1 to 10 times.

[0148] In some embodiments, the population of PDCs is administered 2 to 10 times, 3 to 10 times, 4 to 10 times, 5 to 10 times, 6 to 10 times, 7 to 10 times, 8 to 10 times, or 9 to 10 times.

[0149] In some embodiments, the population of PDCs is administered 2 to 9 times, 3 to 8 times, 4 to 7 times, or 5 to 6 times.

[0150] In some embodiments, the population of PDCs is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. In one embodiment, the population of PDCs is administered 3 times. In a preferred embodiment, the population of PDCs is administered 6 times.

[0151] In some embodiments, at least one blood sample is taken from the subject. It is understood that the blood sample is for further analysis and treatment follow-up, particularly immune monitoring.

[0152] In some embodiments, the at least one blood sample is centrifuged at 300-1000 x g for 10-30 minutes, In some embodiments, the at least one blood sample is mixed with Ficoll-Paque medium prior to centrifugation.

[0153] In certain embodiments, centrifugation forms two or more fractions (e.g., red blood cells, platelets, plasma, peripheral blood mononuclear cells, etc.). In some embodiments, the peripheral blood mononuclear cell (PBMC) fraction is substantially isolated. In some embodiments, the PBMC fraction is substantially purified.

[0154] In some embodiments, the PBMC fraction is frozen. In some embodiments, the PBMC fraction is frozen at -20°C, -80°C, or -196°C, preferably -196°C. In some embodiments, the PBMC fraction is frozen in liquid nitrogen. In a preferred embodiment, the PBMC fraction is contacted with at least one suitable cryoprotectant known in the art.

[0155] In some embodiments, frozen PBMCs are thawed prior to analysis.

[0156] In some embodiments, at least one blood sample, PBMCs, thawed PBMCs and / or purified CD8+ T cells are analyzed, assessed, quantified or screened for CD8+ T cells, preferably activated CD8+ T cells, more preferably ASTC.

[0157] In some embodiments, CD8+ T cells, preferably CD8+CD3+ T cells, are purified from PBMCs or thawed PBMCs by any suitable means known in the art. In a preferred embodiment, CD8+ T cells, preferably CD8+CD3+ T cells, are purified from PBMCs or thawed PBMCs by magnetic-activated cell sorting (MACS).

[0158] In some embodiments, the CD8+ T cells, preferably activated CD8+ T cells, more preferably ASTC, are further subjected to multimer staining. In some embodiments, the multimer staining is performed by contacting the CD8+ T cells, preferably activated CD8+ T cells, more preferably ASTC, with at least one multimer or dextramer comprising one or more HLA-A2 / peptide complexes, wherein the at least one multimer or dextramer is specific for at least one cancer antigen and / or fragment and / or variant thereof.

[0159] In some embodiments, CD8+ T cells, preferably activated CD8+ T cells, more preferably ASTC, are incubated with a fluorescent dye-conjugated antigen-specific dextramer. In some embodiments, the percentage of CD8+ T cells, preferably activated CD8+ T cells, more preferably ASTC, is assessed by flow cytometry. In some embodiments, CD8+ T cells, preferably activated CD8+ T cells, more preferably ASTC, are sorted by any suitable means known in the art, preferably by fluorescence-activated cell sorting (FACS).

[0160] In some embodiments, the viability or live cell marker is used in flow cytometry and / or FACS analysis as known in the art. In a preferred embodiment, the viability or live cell marker is fixable viability stain 510 Dye.

[0161] In some embodiments, at least one blood sample, PBMCs, thawed PBMCs, and / or purified CD8+ T cells are analyzed, assessed, quantified, or screened for a differentiation marker, which in some embodiments is CD45RA and / or CCR7.

[0162] In some embodiments, at least one blood sample, PBMCs, thawed PBMCs, and / or purified CD8+ T cells are analyzed, assessed, quantified, or screened for an activation marker, which in some embodiments is CD25, HLA-DR, and / or CD54.

[0163] In some embodiments, at least one blood sample is analyzed, assessed, quantified, or screened for cancer markers.

[0164] In some embodiments, the methods according to the invention further comprise testing the subject for a cancer marker, preferably testing at least one blood sample for a cancer marker, hi some embodiments, the methods according to the invention further comprise numbering activated CD8+ T cells, preferably ASTC, in the subject.

[0165] In some embodiments, methods according to the invention further comprise assessing and / or quantifying the effectiveness of the treatment, hi some embodiments, methods according to the invention further comprise concluding whether to maintain, continue, adapt, stop, interrupt, discontinue, modify, or resume treatment based on the assessment and / or quantification of the effectiveness of the treatment.

[0166] In some embodiments, the population of PDCs is administered every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks.

[0167] In preferred embodiments, the population of PDCs is administered weekly, every two weeks, or every three weeks.

[0168] In a more preferred embodiment, the population of PDCs is administered weekly.

[0169] Within the scope of the present invention, a period of one week is meant to be understood to expire on the day of the following week having the same name as the day on which the administration took place.

[0170] In some embodiments, the population of PDCs is administered intravenously, subcutaneously, intradermally, intraarterially, intraperitoneally, intramuscularly, and / or intratumorally.

[0171] In a preferred embodiment, the population of PDCs is administered intravenously and / or subcutaneously. In another preferred embodiment, the population of PDCs is administered intravenously and subcutaneously.

[0172] In one embodiment, the population of PDCs is administered intravenously, hi another embodiment, the population of PDCs is administered subcutaneously.

[0173] In another embodiment, the population of PDCs is administered intravenously and subcutaneously.In certain embodiments, the dose that is administered intravenously is the same as the dose that is administered subcutaneously, that is, the dose that is administered by each route is equal to half of the total dose that is administered to the subject.In certain embodiments, the dose that is administered intravenously is different from the dose that is administered subcutaneously, and the total dose that is administered by each route is equal to the total dose that is administered to the subject.

[0174] In some embodiments, the population of PDCs is contained in a solution suitable for human administration, particularly human systemic injection (eg, saline containing 0.9% sodium chloride).

[0175] Non-limiting examples of cancer types include carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Within the scope of the present invention, the cancer is preferably, but not limited to, a carcinoma, such as adenocarcinoma, squamous cell carcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma, etc., or melanoma.

[0176] In some embodiments, the cancer comprises or is selected from the group consisting of lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, squamous cell carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, liver cancer, head and neck cancer, and the like.

[0177] In some embodiments, the cancer is lung cancer or melanoma.

[0178] In a preferred embodiment, the cancer is lung cancer.

[0179] Lung cancer can be differentiated into two major histological categories: small cell lung cancer (SCLC), which accounts for approximately 10-20% of all primary lung cancers, and non-small cell lung cancer (NSCLC).

[0180] Methods for diagnosing lung cancer are known in the art, including but not limited to radiographic screening (X-ray, low-dose helical computed tomography, CT scan), sputum test, bronchoscopy, and lung tissue biopsy.Diagnosis is preferably performed by a medical professional.

[0181] In some embodiments, the subject has a detectable level of a lung cancer marker (e.g., at the protein level of a gene). In some embodiments, the subject has a detectable level of any of the lung cancer markers selected from the group including CYFRA 21-1, carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and squamous cell carcinoma antigen (SCC-Ag).

[0182] In some embodiments, the markers are measured at the protein and / or RNA level.

[0183] In some embodiments, the marker is measured at the protein level. Methods for measuring protein expression are known in the art and include enzyme-linked immunosorbent assay (ELISA), Western blot, dot blot, immunofluorescence, immunochemistry, immunoprecipitation, fluorescence-activated cell sorting (FACS), high-performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS), among others.

[0184] In some embodiments, the marker is measured at the RNA level. Methods for measuring RNA expression are known in the art and include RT-PCR, RT-qPCR, Northern blot, and hybridization techniques, among others.

[0185] In some embodiments, the cancer is small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC).

[0186] In one embodiment, the cancer is NSCLC.

[0187] In some embodiments, the NSCLC comprises or is selected from the group consisting of lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), large cell carcinoma, and bronchial carcinoid tumor, preferably LUAD.

[0188] In another embodiment, the cancer is SCLC.

[0189] In another embodiment, the cancer is melanoma. In some embodiments, the cancer is not melanoma.

[0190] In some embodiments, the cancer, preferably lung cancer, is classified according to the TNM classification, preferably T1a, T1b, T1c, T2a, T2b, T3 size , T3 inv , T3 centr , T3 satell , T4 inv , T4 ipsi , N1 and N2.

[0191] In some embodiments, the cancer is in a metastatic stage.

[0192] In some embodiments, the subject is a smoker or a non-smoker.

[0193] In some embodiments, the method further comprises administering to said subject at least one additional anti-cancer agent.

[0194] In some embodiments, the at least one additional anti-cancer agent is a pharmacologically active molecule, such as a small molecule, or a biologically active peptide / protein. In some embodiments, the at least one additional anti-cancer agent is included in a pharmacological composition approved (e.g., FDA or EMA approved) for the treatment of at least one disease. In some embodiments, the at least one additional anti-cancer agent has undergone at least one preclinical or clinical trial.

[0195] In a preferred embodiment, the at least one additional anti-cancer agent targets at least one gene selected from the list including or consisting of EGFR, ALK, ROS1, BRAF, RET, MET, and HER2.

[0196] Anticancer drugs are known from the state of the art. Non-limiting examples of anticancer drugs include acalabrutinib, alectinib, alemtuzumab, anastrozole, avapritinib, avelumab, belinostat, bevacizumab, bleomycin, blinatumomab, bosutinib, brigatinib, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, copanlisib, cytarabine, daunorubicin, decitabine, dexamethasone, docetaxel, doxorubicin, encorafenib, erdafitinib, etoposide, everolimus, These include exemestane, fludarabine, 5-fluorouracil, gemcitabine, ifosfamide, imatinib mesylate, leuprolide, lomustine, mechlorethamine, melphalan, methotrexate, mitomycin, nelabine, paclitaxel, pamidronate, panobinostat, pralatrexate, prednisolone, ofatumumab, rituximab, temozolomide, topotecan, tositumomab, trastuzumab, vandetanib, vincristine, vorinostat, zanubrutinib, and the like.

[0197] In one embodiment, at least one anti-cancer agent is an immune checkpoint inhibitor. In one embodiment, at least one anti-cancer agent is an inhibitor of programmed cell death protein-1 (PD-1) or an inhibitor of PD-1 ligand (PDL-1). In one embodiment, at least one anti-cancer agent is an anti-PD-1 antibody, preferably selected from the group including or consisting of pembrolizumab, nivolumab, lambrolizumab, dostarimab, and cemiplimab. In one embodiment, at least one anti-cancer agent is pembrolizumab. In one embodiment, at least one anti-cancer agent is an anti-PDL-1 antibody, preferably selected from the group including or consisting of atezolizumab, durvalunab, and avelumab.

[0198] In certain embodiments, at least one anti-cancer agent is to be administered simultaneously or sequentially in combination with a combination for use according to the present invention.

[0199] In some embodiments, the method further comprises administering to said subject at least one adjuvant.

[0200] Adjuvants are known in the art and include, but are not limited to, amorphous metals (e.g., aluminum), altered metals (e.g., aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate, aluminum sulfate hydroxyphosphate, etc.), lipids (e.g., oils; monophosphoryl lipid A, etc.), nucleic acids (e.g., cytosine phosphoguanine), saponins, or other chemicals.

[0201] It will be appreciated that adjuvants are preferentially used prophylactically, i.e. to induce an immune response against an antigen or a fragment or variant thereof, prior to the onset of disease, particularly cancer.

[0202] In some embodiments, the method further comprises assessing the subject's immune background by blood sampling.

[0203] In some embodiments, the step of assessing the immune background of the subject consists of numbering the subject's activated CD8+ T cells, preferably ASTC, and / or measuring their activity, proliferation and / or cytotoxic capacity, preferably oncolytic capacity.

[0204] In some embodiments, the method further comprises assessing the immunogenicity of the subject by at least one blood sampling after vaccination.

[0205] Blood sampling and analysis is described herein.

[0206] The present invention further relates to a therapeutically effective amount of a population of plasmacytoid dendritic cells (PDCs) as described herein above for use in the treatment and / or prevention of cancer, wherein the population of PDCs is for administration or has been administered.

[0207] The present invention further relates to a pharmaceutical composition comprising a therapeutically effective amount of a population of plasmacytoid dendritic cells (PDCs) as described herein above, for treating and / or preventing cancer in a subject in need thereof.

[0208] In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable vehicle or excipient.

[0209] In some embodiments, the pharmaceutically acceptable vehicle or excipient is selected from the group consisting of solvents, diluents, carriers, excipients, dispersion media, coatings, absorption delaying agents, and any combination thereof. The carrier, diluent, solvent, or excipient must be "acceptable" in the sense of being compatible with the population of PDCs and not harmful upon administration to a subject. Typically, the vehicle or excipient does not cause adverse allergic or other untoward reactions when administered to a subject.

[0210] In some embodiments, the populations of PDCs are associated with different pharmaceutically acceptable vehicles or excipients.

[0211] The present invention further relates to a kit comprising a therapeutically effective amount of a population of plasmacytoid dendritic cells (PDCs) as described herein above, for treating and / or preventing cancer in a subject in need thereof.

[0212] The present invention further relates to the use of a therapeutically effective amount of a population of PDCs as described herein above for the manufacture of a medicament for treating and / or preventing cancer.

[0213] In some embodiments, the pharmaceutical product further comprises at least one pharmaceutically acceptable vehicle or excipient as described herein. [Brief explanation of the drawings]

[0214] [Figure 1]Dot plots showing the percentage of ASTC at baseline in patients in cohorts A1 and A2. Blood sampling was performed before treatment. The percentage of ASTC (among CD8+ T cells) was measured by flow cytometry. The antigens studied were NY-ESO-I (NY), MAGE-A3 (M3), MUC-1, survivin (S), and MELAN-A (MEL-A). [Figure 2A] Figure 2A is a composite of graphs showing patient response to standard-dose and high-dose treatment with PDC*-lineage cells. Two cohorts of patients were studied: Cohort A1, treated with standard-dose PDC*-lineage cells, and Cohort A2, treated with high-dose PDC*-lineage cells. Blood samples were taken before and after treatment. The percentage of ASCT (among CD8+ T cells) was measured by flow cytometry. The antigens NY-ESO-I (NY), MAGE-A3 (M3), MUC-1, survivin (S), and MELAN-A (MEL-A) were studied. Figure 2A is an exemplary dot plot showing the percentage of ASCT for three positive patients (P6, P7, and P9) from Cohort A1. Figure 2B is an exemplary dot plot showing the percentage of ASCT for seven positive patients (P02, P04, P06, P09, P10, P11, and P13) from Cohort A2. Figure 2C shows a dot plot summarizing the percentage of ASTCs from each cohort after treatment. Each dot represents an ASTC proliferation. If several proliferations were observed in the same patient, all proliferations are shown. Figure 2D shows a histogram representing the percentage of patients with an immune response. Non-positive patients are not shown. [Figure 2B]Figure 2A is a composite of graphs showing patient response to standard-dose and high-dose treatment with PDC*-lineage cells. Two cohorts of patients were studied: Cohort A1, treated with standard-dose PDC*-lineage cells, and Cohort A2, treated with high-dose PDC*-lineage cells. Blood samples were taken before and after treatment. The percentage of ASCT (among CD8+ T cells) was measured by flow cytometry. The antigens NY-ESO-I (NY), MAGE-A3 (M3), MUC-1, survivin (S), and MELAN-A (MEL-A) were studied. Figure 2A is an exemplary dot plot showing the percentage of ASCT for three positive patients (P6, P7, and P9) from Cohort A1. Figure 2B is an exemplary dot plot showing the percentage of ASCT for seven positive patients (P02, P04, P06, P09, P10, P11, and P13) from Cohort A2. Figure 2C shows a dot plot summarizing the percentage of ASTCs from each cohort after treatment. Each dot represents an ASTC proliferation. If several proliferations were observed in the same patient, all proliferations are shown. Figure 2D shows a histogram representing the percentage of patients with an immune response. Non-positive patients are not shown. [Figure 2C]Figure 2A is a composite of graphs showing patient response to standard-dose and high-dose treatment with PDC*-lineage cells. Two cohorts of patients were studied: Cohort A1, treated with standard-dose PDC*-lineage cells, and Cohort A2, treated with high-dose PDC*-lineage cells. Blood samples were taken before and after treatment. The percentage of ASCT (among CD8+ T cells) was measured by flow cytometry. The antigens NY-ESO-I (NY), MAGE-A3 (M3), MUC-1, survivin (S), and MELAN-A (MEL-A) were studied. Figure 2A is an exemplary dot plot showing the percentage of ASCT for three positive patients (P6, P7, and P9) from Cohort A1. Figure 2B is an exemplary dot plot showing the percentage of ASCT for seven positive patients (P02, P04, P06, P09, P10, P11, and P13) from Cohort A2. Figure 2C shows a dot plot summarizing the percentage of ASTCs from each cohort after treatment. Each dot represents an ASTC proliferation. If several proliferations were observed in the same patient, all proliferations are shown. Figure 2D shows a histogram representing the percentage of patients with an immune response. Non-positive patients are not shown. [Figure 2D]Figure 2A is a composite of graphs showing patient response to standard-dose and high-dose treatment with PDC*-lineage cells. Two cohorts of patients were studied: Cohort A1, treated with standard-dose PDC*-lineage cells, and Cohort A2, treated with high-dose PDC*-lineage cells. Blood samples were taken before and after treatment. The percentage of ASCT (among CD8+ T cells) was measured by flow cytometry. The antigens NY-ESO-I (NY), MAGE-A3 (M3), MUC-1, survivin (S), and MELAN-A (MEL-A) were studied. Figure 2A is an exemplary dot plot showing the percentage of ASCT for three positive patients (P6, P7, and P9) from Cohort A1. Figure 2B is an exemplary dot plot showing the percentage of ASCT for seven positive patients (P02, P04, P06, P09, P10, P11, and P13) from Cohort A2. Figure 2C shows a dot plot summarizing the percentage of ASTCs from each cohort after treatment. Each dot represents an ASTC proliferation. If several proliferations were observed in the same patient, all proliferations are shown. Figure 2D shows a histogram representing the percentage of patients with an immune response. Non-positive patients are not shown. [Figure 3A]Figure 3A is a composite graph showing patient responses following standard-dose and high-dose treatment with PDC*-lineage cells. Two cohorts of patients were studied: Cohort A1, treated with standard-dose PDC*-lineage cells, and Cohort A2, treated with high-dose PDC*-lineage cells. Blood sampling was performed 1 week (V7), 4 weeks (V8), and 10 weeks (V9) after the last dose of PDC*-lineage cells. The percentage of ASTC (among CD8+ T cells) was measured by flow cytometry. The antigens NY-ESO-I (NY), MAGE-A3 (M3), MUC-1, survivin (S), and MELAN-A (MEL-A) were studied. Figure 3A is an exemplary dot plot showing the effect of standard-dose PDC*lung01 on the percentage of ASTC in three positive patients (P6, P7, and P9) from Cohort A1. Figure 3B is an exemplary dot plot showing the effect of high-dose PDC*lung01 combination on the rate of ASTC in seven positive patients (P02, P04, P06, P09, P10, P11, and P13) in cohort A2. Non-positive patients are not shown. [Figure 3B] Figure 3A is a composite graph showing patient responses following standard-dose and high-dose treatment with PDC*-lineage cells. Two cohorts of patients were studied: Cohort A1, treated with standard-dose PDC*-lineage cells, and Cohort A2, treated with high-dose PDC*-lineage cells. Blood sampling was performed 1 week (V7), 4 weeks (V8), and 10 weeks (V9) after the last dose of PDC*-lineage cells. The percentage of ASTC (among CD8+ T cells) was measured by flow cytometry. The antigens NY-ESO-I (NY), MAGE-A3 (M3), MUC-1, survivin (S), and MELAN-A (MEL-A) were studied. Figure 3A is an exemplary dot plot showing the effect of standard-dose PDC*lung01 on the percentage of ASTC in three positive patients (P6, P7, and P9) from Cohort A1. Figure 3B is an exemplary dot plot showing the effect of high-dose PDC*lung01 combination on the rate of ASTC in seven positive patients (P02, P04, P06, P09, P10, P11, and P13) in cohort A2. Non-positive patients are not shown. [Example]

[0215] The present invention is further illustrated by the following examples. Example 1

[0216] Higher potency of high doses of PDC* lineage cells to induce ASTC proliferation.

[0217] material and method Preparation of tumor peptide-loaded PDC* cells

[0218] PDC* cells were separately loaded with distinct synthetic peptides encoded by lung tumor antigens, namely NY-ESO-1 (NY), MAGE-A3 (M3), MUC1, and survivin (S), or a peptide derived from the Melan-A antigen (MelA), used as a positive immunogenicity control. Briefly, PDC* cells (1,000,000 / mL) were incubated with 10 μM of each peptide at 37°C for 3 hours. Tumor peptides were obtained from PolyPeptide Laboratories, Inc. The characteristics of the peptides are detailed in Table 1. After loading, cells were washed, irradiated, and stored frozen in cryotubes until use.

[0219] [Table 1]

[0220] PDC*-lineage cells were administered as a single agent (Cohort A1) or in addition to anti-PD-1 (Cohort B1). Patients in Cohort A1 were treated with 2 million PDC*-lineage cells per tumor antigen loading (i.e., 2 million PDC*-lineage cells loaded with NY, 2 million PDC*-lineage cells loaded with M3, etc.) at each of six weekly treatment visits, with 1 million administered subcutaneously and 1 million administered intravenously. Patients in Cohort A2 were also treated with 20 million PDC*-lineage cells per tumor antigen loading (i.e., 10 million administered subcutaneously and 10 million administered intravenously) at each of six weekly treatment visits.

[0221] Patients in cohort A1 were different from patients in cohort A2. For each cohort, patients were numbered P01, P02, P03, etc.

[0222] To assess the basal percentage of antigen-specific T cells (ASTCs) in each patient, a pre-experimental blood sampling analysis was performed. Peripheral blood samples were collected 1 week (V7), 4 weeks (V8), and 10 weeks (V9) after the last PDC* administration. PBMCs were purified from patient blood using Ficoll-Hypaque density gradient centrifugation (Lymphocyte Separation Medium, Eurobio) and stored frozen until analysis.

[0223] Detection of antigen-specific CD8+ T cells The percentage of ASTC proliferation was assessed by flow cytometry after each blood sampling.

[0224] After thawing the samples, the PBMCs were subjected to red blood cell lysis. The samples were then subjected to CD8+ T cell purification using MACS technology. Briefly, magnetic labeling was performed as follows: the PBMC pellet was thawed at 10 × 10 6 Resuspend 10 x 10 cells in 40 µl purification buffer for every 10 x 10 total PBMCs 6Each 10 x 10 total PBMCs were incubated with 10 μl of biotin-antibody cocktail for 5 minutes. 6 For each 100 μL of total PBMCs, 30 μL of purification buffer and 20 μL of CD8+ T cell microbead cocktail were added and incubated at 4°C for 15 minutes. Magnetic separation of CD8+ T cells was then performed as follows: the PBMC suspension was applied to each LS column, and the flow-through containing unlabeled cells (representing enrichment for CD8+ T cells) was collected. To maximize CD8+ T cell recovery, the columns were then washed three times with purification buffer. The magnetically labeled cells (non-CD8+ T cells) were eluted from the column with purification buffer.

[0225] The sample enriched for CD8+ T cells was then subjected to dextramer labeling. Briefly, cells were centrifuged and the cell pellet was then diluted to 1×10 6 The cells were then resuspended in PBS-2% FBS-2 mM EDTA + 10 μM biotin at a concentration of 1.00 × 10 viable cells / ml. 6After incubation with 0.5 μl of fixable viability stain 510 Dye for 20 min, the cells were washed and incubated with fluorescent dye-conjugated antigen-specific dextramers (PE dextramer MAGE-A3 Cat: WB3415-PE; PE dextramer Survivin Cat: WB2167-PE; FITC dextramer NY-ESO-1 Cat: WB3247-FITC; APC dextramer Multi-MAGE Cat: WB3880-APC; PE dextramer Melan-A Cat: WB2162-PE; Immudex, Denmark) for 20 min in the dark at room temperature. After washing, BUV737 mouse anti-human CD3 antibody (BD Biosciences, Cat: 612752), BV421 mouse anti-human CD8 antibody (BD Biosciences, Cat: 562428), BUV395 mouse anti-human CD45RA antibody (BD Biosciences, Cat: 740315), and BV785 mouse anti-human CCR7 antibody (BioLegend, Cat: 353230) were added and incubated for 20 minutes at 4°C in the dark. The cells were then washed, resuspended in FACS syringe solution, and fluorescence was detected using a flow cytometer (BD FACS Aria III). The frequency of multimer-positive cells was measured in the CD8-positive live single-cell population of lymphocytes.

[0226] Patient PBMCs and cells collected at the end of the coculture were resuspended in PBS containing 2% decomplemented FCS (Gibco, Life Technologies, France) and incubated with fluorescent dye-conjugated antigen-specific multimers, either tetramers (ITag, Beckman Coulter, Villepinte) or dextramers (Immudex, Denmark), for 20 min in the dark at room temperature.

[0227] After washing, BV421-conjugated anti-CD3 antibody (BD Biosciences, Le Pont de Claix, France) and PerCP-Cy5.5-conjugated anti-CD8 antibody (BD Biosciences, Le Pont de Claix, France) were added and incubated for 20 minutes in the dark at 4°C. Viability dye (Live and Dead, Fisher Scientific, Illkirch, France) was added to the antibody mix. After washing and resuspending the cells in FACS lysis solution (BD Biosciences, #349202), fluorescence was acquired using a flow cytometer (BD FACS Canto II) and analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA). The frequency of multimer-positive cells was measured in the CD8-positive live single-cell population of lymphocytes, as described in Figure 2.

[0228] The quantification limits for ASTC were set at 0.005% of total gated CD8+ T cells for MAGE-A3-specific, survivin-specific, and Melan-A-specific CD8+ T cells and 0.003% for other specific CD8+ T cells.

[0229] result First, the frequency of ASTC at baseline was determined for cohorts A1 and A2. The results showed that the basal frequency of ASTC in A1 and A2 was similar (Figure 1 and Table 2).

[0230] [Table 2]

[0231] Patients could then be evaluated for immunogenicity assessment of PDC* lineage cells at standard versus high doses.

[0232] A patient is positive if the percentage of ASTC of at least one of the seven peptides increases two-fold (taking into account the defined limit of detection (LOQ)) between baseline and V7 or V8 or V9 (i.e., corresponding to the post-treatment blood sampling).

[0233] The results revealed that treatment with high-dose PDC* cells resulted in a greater number of patients demonstrating immune responses. Indeed, the number of positive patients was higher in Cohort A2 (7 / 11, i.e., 63% positive patients) than in Cohort A1 (3 / 6, i.e., 50% positive patients). Furthermore, the magnitude of this response was greater in Cohort A2, which demonstrated responses to four lung antigens (M1, M3, NY, and S) compared with Cohort A1, which demonstrated responses to only two lung antigens (M3 and S) (Figure 2A, Figure 2B, Figure 2C, and Figure 2D).

[0234] The same results are shown taking into account the delay between sampling (Figure 3A corresponds to Table 3, and Figure 3B corresponds to Table 4). The tabulated results for antigen responses in positive patients are summarized in Table 5.

[0235] Taken together, these results demonstrated that a high dose of PDC* lineage cells enhanced ATSC responses compared with a standard dose of PDC*lung01.

[0236] [Table 3]

[0237] [Table 4]

[0238] [Table 5]

Claims

1. 1. A method of treating and / or preventing cancer in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a population of plasmacytoid dendritic cells (PDCs), wherein said therapeutically effective amount is at least 5,000,000 cells.

2. 10. The method of claim 1, wherein the population of PDCs is contacted with at least one cancer antigen and / or fragment and / or variant thereof.

3. 3. The method of claim 2, wherein the therapeutically effective amount is per cancer antigen and / or fragment and / or variant thereof.

4. The at least one cancer antigen and / or fragment and / or variant thereof is selected from the group consisting of MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, CAMEL (CTAG2), GLULD1 (LGSN), HER2 (ERBB2), MUC1, survivin (BIRC5), NY-ESO-1, MULTI-MAGE, NY-BR-1, cyclin D1 (CCND1), PD-L1, CEA, EPCAM, IDO, and LY-6K. , MUC5AC and 5T4 (TPBG), preferably wherein said at least one cancer antigen and / or fragment and / or variant thereof comprises or is selected from the group consisting of MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A9, MUC1, survivin (BIRC5), NY-ESO-1, CAMEL, MULTI-MAGE, 5T4 (TPBG) and CCND1.

5. 10. The method of claim 1, wherein the population of PDCs is administered at least once, preferably at least three times, and more preferably at least six times.

6. 10. The method of claim 1, wherein the population of PDCs is administered 1 to 10 times.

7. 2. The method of claim 1, wherein the cancer comprises or is selected from the group consisting of lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, squamous cell carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatic cancer, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, liver cancer, head and neck cancer, and the like.

8. 10. The method of claim 1, wherein the cancer is lung cancer or melanoma.

9. 10. The method of claim 1, wherein the cancer is small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC).

10. 10. The method of claim 1, wherein the cancer is NSCLC.

11. 10. The method of claim 1, further comprising administering to the subject at least one additional anti-cancer agent.

12. 10. The method of claim 1, further comprising administering at least one adjuvant to the subject.

13. 10. The method of claim 1, further comprising assessing the subject's immune background by blood sampling.

14. 10. The method of claim 1, further comprising assessing the immunogenicity of the subject by at least one blood sampling after vaccination.

15. A population of plasmacytoid dendritic cells (PDCs) for use in the treatment and / or prevention of cancer, the population of PDCs being for or to be administered in a therapeutically effective amount of at least 5,000,000 cells.

16. A pharmaceutical composition comprising a population of plasmacytoid dendritic cells (PDCs) in a therapeutically effective amount of at least 5,000,000 cells for treating and / or preventing cancer in a subject in need thereof.

17. A kit comprising a therapeutically effective amount of a population of plasmacytoid dendritic cells (PDCs) of at least 5,000,000 cells for treating and / or preventing cancer in a subject in need thereof.

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